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Increase 5' homogeneity of in vitro transcribed RNA

Structural investigations by NMR or X-ray crystallography require a homogenous RNA preparation. However, in vitro transcription-based RNA synthesis with T7 RNA polymerase produces heterogenous RNA at both 5' and 3' ends especially if prepared under the commonly used class III G-initiating phi6.5 promotor.

The combination of

  • a modified T7 RNA polymerase version (T7 P&L RNA polymerase) with proline 266 replaced by leucine (P266L)
and
  • a class II A-initiating phi2.5 promotor (5'-TAATACGACTCACTATTANN...-3' or 5'-TAATACGACTCACTATAANN...-3')
has been associated with markedly increased 5' homogeneity of RNA transcripts[1-3].

Table 1: Overview on available T7 P&L RNA Polymerase Kits and related single substances

Product Cat.-No. Amount Price Application Average yield / react.*
HighYield T7 P&L RNA Synthesis Kit RNT-201 50 react. 195,00 € Optimized kit for high yield RNA synthesis 140 - 160 µg
T7 P&L RNA Polymerase HC RNT-018 50 µl (2 µg/µl) 365,00 € Single enzyme for individual assay set-ups n/a
HighYield T7 P&L RNA NMR Kit (5F-UTP) RNT-202-S 15 react. 395,00 € Optimized kit for preparation of 5-Fluoro-modified RNA 80 µg
RNT-202-L 50 react. 895,00 €
5-Fluoro-UTP (5F-UTP) NU-1115S 10 µl (100 mM) 123,24 € Single nucleotide for individual assay set-up n/a
NU-1115L 5x 10 µl (100 mM) 360,95 €
HighYield T7 P&L RNA NMR Kit (2F-ATP) RNT-203-S 15 react. 535,00 € Optimized kit for preparation of 2-Fluoro-modified RNA 80 µg
RNT-203-L 50 react. 1.140,00 €
2-Fluoro-ATP (2F-ATP) NU-145S 10 µl (100 mM) 180,47 € Single nucleotide for individual assay set-up n/a
NU-145L 5x 10 µl (100 mM) 523,36 €
* reaction conditions according to corresponding datasheet

Figure 1: T7 P&L RNA polymerase possesses similar activity as wildtype enzyme.
Figure 1: T7 P&L RNA polymerase possesses similar activity as wildtype enzyme.
RNA synthesis has been monitored in real time with a fluorescence aptamer assay (2 µg each T7 RNA Polymerase, 7.5 mM NTPs, 1 µg F30-2x Broccoli aptamer DNA template, 0.1 mM DFHBI-1T, 37 °C, read out: Tecan plate reader).

Questions or inquiries?

Dr. Barbara Zschoernig

Please contact Barbara with all questions or inquiries you may have!

Selected References:

[1] Coleman et al. (2004) Superior 5‘ homogeneity of RNA from ATP-initiated transcription under T7 phi 2.5 promoter. Nucleic Acids Research 32 (1):e14.
[2] Guillerez et al. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance. Natl. Acad. Sci. U.S.A. 102:5958.
[3] Salvail-Lacoste et al. (2018) Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags. RNA 19:1003.